Sustained release of microcrystalline peptide suspensions

ABSTRACT

The invention relates to a microcrystalline aqueous suspension of a peptide salt selected from the group consisting of Ac—D—Nal—D—Cpa—D—Pal—Ser—Tyr—D—Hci—Leu—Ilys—Pro—D—Ala—NH 2 trifluoroacetate and Ac—D—Nal—D—Cpa—D—Pal—Ser—Tyr—D—Hci—Leu—Ilys—Pro—D—Ala—NH 2  sulfate. The invention also relates to methods of preparing the suspension, lyophilized compositions formed from the suspensions, and sustained release formulations that include the suspensions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No. 10/080,130, filed Feb. 19, 2002, which claims priority to U.S. Provisional Application No. 60/317,616, filed Sept. 6, 2001, the entire disclosure of each of which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

There is frequently a need to deliver biologically active peptides to animals and humans in formulations providing a sustained release of the active principle. Such formulations may be provided by incorporating the active principle in biodegradable and biocompatible polymers in the form of microcapsules, microgranules or implantable rods, or alternatively using mechanical devices such as micropumps or non-biodegradable containers. If the peptide is highly soluble in aqueous media, it can be formulated as a complex with non-degradable polymers such as cellulose derivatives, or mixed with polymer solutions, which form a gel upon parenteral injection, from which the active peptide is slowly released.

All the above-mentioned formulations have drawbacks and limitations, such as the large volume of suspending fluids or the need to remove the non-degradable device. In the case of gel forming peptides, there is frequently a problem of bioavailability, which interferes with the desired sustained action of the active principle.

Some of the problems due to physico-chemical aspects of peptides have been described in an article by R. Deghenghi “Antarelix” in Treatment with GnRH Analogs: Controversies and Perspectives”, edited by M. Filicori and C. Flamigni, The Parthenon Publishing Group, New York and London 1996, pages 89-91. Additional problems were illustrated by J. Rivier “GnRH analogues towards the next millennium” in GnRH Analogues, edited by B. Lunenfeld, The Parthenon Publishing Group, New York and London 1999, pages 31-45 and by other workers such as M. F. Powell et al. “Parenteral Peptide Formulations: Chemical and Physical Properties of Native LHRH and Hydrophobic Analogues in Aqueous Solution” in Pharmaceutical Research, Vol. 8, 1258-1263 (1991).

Accordingly, there is a need for new formulations and methods of administration that avoid these problems, and this need is addressed by the present invention.

SUMMARY OF THE INVENTION

The present invention relates to a microcrystalline aqueous suspension of a peptide salt selected from the group consisting of Ac—D—Nal—D—Cpa—D—Pal—Ser—Tyr—D—Hci—Leu—Ilys—Pro—D—Ala—NH2trifluoroacetate and Ac—D—Nal—D—Cpa—D—Pal—Ser—Tyr—D—Hci—Leu—Ilys—Pro—D—Ala—NH₂ sulfate.

In a preferred embodiment, the peptide salt is Ac—D—Nal—D—Cpa—D—Pal—Ser—Tyr—D—Hci—Leu—Ilys—Pro—D—Ala—NH2trifluoroacetate and is suspended in an aqueous medium at a concentration of equal to or greater than 25 mg/mL. The suspension may include other ingredients, for example, an isotonic agent, such as mannitol.

In another preferred embodiment, the peptide salt is Ac—D—Nal—D—Cpa—D—Pal—Ser—Tyr—D—Hci—Leu—Ilys—Pro—D—Ala-NH2sulfate and is suspended in an aqueous medium at a concentration of equal to or greater than 25 mg/mL. The suspension typically also includes an isotonic agent, such as mannitol.

The suspension can also include a pharmaceutically acceptable excipient. Typically, the peptide salt is at least partially in the form of microcrystals in the form of needles having a particle size of from about 1 μm to 150 μm.

The present invention also relates to a method of preparing the suspension that includes associating the peptide with a counter-ion in an amount and at a molar ratio that are sufficient to provide a fluid, milky microcrystalline aqueous suspension that includes the peptide salt without formation of a gel. Preferably, the counter ion is a trifluoroacetate and the peptide salt that is formed is Ac—D—Nal—D—Cpa—D—Pal—Ser—Tyr—D—Hci—Leu—Ilys—Pro—D—Ala—NH₂trifluoroacetate. In another preferred embodiment, the counter ion is a sulfate and the peptide salt that is formed is Ac—D—Nal—D—Cpa—D—Pal—Ser—Tyr—D—Hci—Leu—Ilys—Pro—D—Ala—NH₂ sulfate. The method also generally includes lyophilizing the suspension to prepare a lyophilized composition.

The present invention further relates to a lyophilized composition that includes a dried suspension obtained by the method. In addition, the present invention relates to a method of preparing a microcrystalline aqueous suspension that includes adding water or buffer with mixing to the lyophilized composition.

The present invention also encompasses a sustained release formulation that includes the suspension, which, when administered to a subject, releases the peptide salt in vivo over a period of at least two weeks. The peptide salt is preferably Ac—D—Nal—D—Cpa—D—Pal—Ser—Tyr—D—Hci—Leu—Ilys—Pro—D—Ala—NH₂trifluoroacetate or Ac—D—Nal—D—Cpa—D—Pal—Ser—Tyr—D—Hci—Leu—Ilys—Pro—D—Ala—NH2sulfate.

In one embodiment, the suspension is a fluid, milky microcrystalline aqueous suspension. The present invention also relates to a lyophilized composition formed from a lyophilized suspension, which includes the suspension. The peptide salt is Ac—D—Nal—D—Cpa—D—Pal—Ser—Tyr—D—Hci—Leu—Ilys—Pro—D—Ala—NH2trifluoroacetate, and the suspension further includes an isotonic agent. In addition, the present invention relates to a method of preparing a fluid, milky microcrystalline aqueous suspension of Ac—D—Nal—D—Cpa—D—Pal—Ser—Tyr—D—Hci—Leu—Ilys—Pro—D—Ala—NH2trifluoroacetate that includes adding water or buffer with mixing to the lyophilized composition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph which illustrates the pharmacodynamic effect (testosterone suppression) obtained by subcutaneous injection in rats of a suspension of Teverelix trifluoroacetate according to the invention; and

FIG. 2 is a graph which illustrates the sustained release of the peptide Teverelix for several weeks in rats injected with the suspension of Teverelix trifluoroacetate according to the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is directed to the unexpected discovery that certain peptides can be prepared or associated with various counter-ions and simply formulated to provide desirable suspensions of the peptide, which suspensions are highly useful for administering the suspension by injection. In particular, a fluid, milky, stable microcrystalline suspension of the peptide is obtained without formation of a gel that would interfere with the handling of the suspension or the bioavailability of the peptide after injection.

The peptide that is to be utilized in the present suspension can be any one of a variety of well known bioactive peptides or peptide analogues which mimic such peptides. Advantageously, these peptides are formulated to obtain a delayed and sustained release of the peptide after injection. While any peptide can be utilized in this invention, those peptides or peptidomimetics having between 3 and 45 amino acids have been found to be the most suitable. In particular, representative peptides or peptidomimetics are well known to those of ordinary skill in the art and need not be exhaustively mentioned here. Typical examples include GnRH analogues and antagonists, as well as somatostatin and analogues thereof. Specific peptides include Azaline B, Abarelix, Antide, Ganirelix, Cetrorelix, FE 200486, Vapreotide, Octreotide, Lanreotide and SOM-230. These peptides have between 6 and 12 amino acids and are synthetically made to mimic the biological activity of GnRH or somatostatin. The examples mention further preferred peptides.

It has been found that certain counter-ions are highly preferred for obtaining sustained release of the peptide. Suitable counter-ions are those which are strong proton donors. While many compounds are well known to provide this function, the most preferred are strong acids. Sulfuric acid, a well known commodity, is quite useful for this purpose, as are other strong inorganic acids. Sulfuric is preferred due to its ready formation of suitable sulfate salts with the peptides of the invention. Strong organic acids can also be used as counter-ions. These acids include sulfonic acids, such as trifluoromethanesulfonic acid and benzene sulfonic acid. Others, such as trifluoroacetic acid or other fluorinated acids can be used if desired.

The amount of counter-ion is preferably that which is in excess of what is necessary to form a stoichiometric salt of the peptide. The amount of counter-ion is typically at least 1.6 mol acid/mole peptide and preferably 2 mol/mol or greater. While no upper limit has been determined, the amount can be as high as 10 mol/mol. In addition, the injectable suspension should be concentrated to obtained the most desirable release profiles. By concentrated, we mean that the amount of peptide should be above 2.5% by weight of the overall formulation. This is conveniently achieved by adding to water or a buffer solution at least 25 mg/mL of the peptide. Amounts of as high as 100 mg/mL can be used, and these suspensions can also contain other additives. In addition to conventional pharmaceutically acceptable excipients, an isotonic agent, such as mannitol, can be included for its known purpose. Other usual pharmaceutical additives can be included, as desired.

The suspensions can be dried by freeze-drying or spray drying to form lyophilized compositions that can be stored as is and later reconstituted with sterile water or buffer solutions when an injectable formulation is to be prepared. These lyophilized compositions can be stored for relatively long periods of time prior to use. Also they can be easily sterilized and handled until the time when they are to be reconstituted.

An additional advantage of this discovery is the small volume of such suspensions, allowing parenteral injections through a fine needle and thus improving the local tolerance of the injected material. Furthermore, the material can also be used for the local treatment of diseased tissues, e.g., brachytherapy. The peptide is partially or totally in the microcystalline form having a particle size of between about 1 and 150 μm, and preferably between about 5 and 25 μm. These small particles easily pass through the injection needle. In such injections, the amount of peptide ranges from about 0.1 to 5 mg per kg body weight of the mammal or human to which the suspension is to be administered.

A specific discovery was that a highly concentrated aqueous suspension of the peptide of the formula Ac—D—Nal—D—pClPhe—D—Pal—Ser—Tyr—D—Hci—Leu—Lys(iPr)—Pro—D—Ala—NH₂ (Teverelix, a GnRH antagonist) as a trifluoroacetate (TFA) or sulfate salt does not, as might be expected by its hydrophobic character, form a gel but instead forms a microcrystalline milky suspension which is easy to inject parenterally in animals or humans, and which releases the active principle over several weeks (see FIGS. 1 and 2). Such behavior is not elicited by other salts such as the acetate, which result in the expected, but unwanted, formation of gels with poor bioavailability in vivo.

The invention thus represents a simple and elegant solution to the problem of how to suppress gelation of peptide salts while obtaining a prolonged sustained delivery of peptides in the form of highly concentrated suspensions.

EXAMPLES Example 1

200 μL of 5% mannitol were added to approximately 15 mg of the LHRH antagonist Teverelix trifluoroacetate. The mixture was stirred using vortex during one minute and a flowing milky pearly suspension was obtained. The suspension is made of microcrystals of about 10 μm length. Microcrystals may clump together to form urchin like structures. The suspension was injected in rats (1 mg) sub-cutaneously and provided the pharmacodynamic effect of testosterone suppression for more than 45 days (FIG. 1). The pharmacokinetic analysis showed a sustained release of the peptide for several weeks (FIG. 2).

Example 2

200 μL of water were added to approximately 15 mg of the LHRH antagonist Teverelix trifluoroacetate. The mixture was stirred using vortex during one minute and a flowing milky pearly suspension was obtained.

Example 3

200 μL of water were added to approximately 15 mg of the LHRH antagonist Teverelix acetate. The mixture was stirred using vortex during one minute and a transparent gel was obtained. The addition of 20 μL of TFA (3 mols/mol) to the gel resulted in the formation of a fluid, flowing milky pearly suspension.

Example 4

200 μL of 100 mM TFA were added to approximately 15 mg of the LHRH antagonist Teverelix acetate (2 mols/mol) to obtain a flowing milky suspension. In addition, mixing 200 μL of 75 mM TFA with approximately 15 mg of the LHRH antagonist Teverelix acetate (1.5 mol/mol) resulted in a transparent gel being obtained after mixing. In another study, 100 μL of TFA of various concentrations were added to 7.5 mg of the LHRH antagonist Teverelix acetate, with the TFA/Teverelix molar ratio ranging from 1 to 3. A flowing milky suspension was obtained with molar ratios of 1.6, whereas gels were obtained at other molar ratios.

Example 5

200 μL of 150 mM TFA were added to amounts of the LHRH antagonist Teverelix acetate ranging from 5 to 30 mg (concentration ranging from 25 to 150 mg/mL). A flowing milky suspension was obtained with concentrations up to 100 mg/mL.

Example 6

200 μL of 150 mM TFA were added to approximately 15 mg of the LHRH antagonist Teverelix acetate (3 mols/mol) and a flowing milky suspension was obtained after mixing. The suspension was freeze-dried overnight. 200 μL of water or 5% mannitol were added to the lyophilisate and a flowing milky suspension was obtained after mixing and reconstitution.

Example 7

1 mL of 150 mM TFA were added to approximately 75 mg of the LHRH antagonist Teverelix acetate (3 mols/mol) and a flowing milky suspension was obtained after mixing. The suspension was freeze-dried overnight. 1 mL of water and 0.2 M acetate buffer pH 4.0 were added to the lyophilisate and a flowing milky suspension was obtained after mixing and reconstitution. These suspensions were stable for at least 3 days at room temperature.

Example 8

100 μL of a 250 mM H₂SO₄ were added to 7.5 mg of the LHRH antagonist Teverelix acetate (5 mols/mol) and a flowing milky suspension was obtained after several hours. The suspension is made of microcrystals of about 100 μm length. Microcrystals may assemble together to form urchin like structures. The suspension was freeze-dried overnight. 100 μL of water or 5% mannitol were added to the lyophilisate and a flowing milky suspension was obtained after mixing and reconstitution.

Example 9

100 μL of a 150 mM trifluoromethane sulfonic acid solution were added to 7.5 mg of Teverelix acetate to obtain a free flowing milky suspension after mixing.

Example 10

100 μL of a 150 mM solution of benzenesulfonic acid were added to 7.5 mg Teverelix hydrochloride to give after a mixing a free flowing suspension.

Example 11

100 μL of a 200 mM solution of trifluoroacetic acid solution were added to 2.5 mg of Cetrorelix acetate to obtain a milky free flowing suspension.

Example 12

Free flowing suspensions were obtained by adding 100 μL of a 150 mM trifluoroacetic acid solution to 7.5 mg each of the following somatostatin analogues: D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Trp-NH₂ D-2Me-Trp-c[Cys-Phe-D-Trp-Lys-Thr-Cys]-Trp(2Me)- NH₂ D-Nal-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Trp(2Me)-NH₂ D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Trp(2Me)-NH₂

Example 13

100 μL of a 5% mannitol—water solution were added to approximately 5 mg of the somatostatin analog known under the designation SOM 230, i.e., ETD-carboxy-c[Hyp—Phg—D—Trp—Lys—Tyr(Bzl)—Phe], as the trifluoroacetate salt. A milky free flowing suspension was thus obtained. 

1. A microcrystalline aqueous suspension of a peptide salt selected from the group consisting of Ac—D—Nal—D—Cpa—D—Pal—Ser—Tyr—D—Hci—Leu—Ilys—Pro—D—Ala—NH₂trifluoroacetate and Ac—D—Nal—D—pa—D—Pal—Ser—Tyr—D—Hci—Leu—Ilys—Pro—D—Ala—NH₂sulfate.
 2. The suspension of claim 1, wherein the peptide salt is Ac—D—Nal—D—Cpa—D—Pal—Ser—Tyr—D—Hci—Leu—Ilys—Pro—D—Ala—NH₂trifluoroacetate.
 3. The suspension of claim 1, wherein the peptide salt is Ac—D—Nal—D—Cpa—D—Pal—Ser—Tyr—D—Hci—Leu—Ilys—Pro—D—Ala—NH₂sulfate.
 4. The suspension of claim 1, wherein the peptide salt is Ac—D—Nal—D—Cpa—D—Pal—Ser—Tyr—D—Hci—Leu—Ilys—ProD—Ala—NH₂ trifluoroacetate and is suspended in an aqueous medium at a concentration of equal to or greater than 25 mg/mL.
 5. The suspension of claim 4, further comprising an isotonic agent.
 6. The suspension of claim 5, wherein the isotonic agent is mannitol.
 7. The suspension of claim 1, wherein the peptide salt is Ac—D—Nal—D—Cpa—D—Pal—Ser—Tyr—D—Hci—Leu—Ilys—Pro—D—Ala—NH₂ sulfate and is suspended in an aqueous medium at a concentration of equal to or greater than 25 mg/mL.
 8. The suspension of claim 7, further comprising an isotonic agent.
 9. The suspension of claim 8, wherein the isotonic agent is mannitol.
 10. The suspension of claim 1, further comprising a pharmaceutically acceptable excipient.
 11. The suspension of claim 1, wherein the peptide salt is at least partially in the form of microcrystals in the form of needles having a particle size of from about 1 μm to 150 μm.
 12. A method of preparing the suspension of claim 1 which comprises, associating the peptide with a counter-ion in an amount and at a molar ratio that are sufficient to provide a fluid, milky microcrystalline aqueous suspension comprising the peptide salt without formation of a gel.
 13. The method of claim 12, wherein the counter ion is a trifluoroacetate and the peptide salt that is formed is Ac—D—Nal—D—Cpa—D—Pal—Ser—Tyr—D—Hci—Leu—Ilys—Pro—D—Ala—NH₂trifluoroacetate.
 14. The method of claim 12, wherein the counter ion is a sulfate and the peptide salt that is formed is Ac—D—Nal—D—Cpa—D—Pal—Ser—Tyr—D—Hci—Leu—Ilys—Pro—D—Ala—NH₂sulfate.
 15. The method of claim 12 which further comprises, lyophilizing the suspension of claim 1 to prepare a lyophilized composition.
 16. A lyophilized composition comprising a dried suspension obtained by the method of claim
 15. 17. A method of preparing a microcrystalline aqueous suspension which comprises adding water or buffer with mixing to the lyophilized composition of claim
 16. 18. A sustained release formulation comprising the suspension of claim 1 which, when administered to a subject, releases the peptide salt in vivo over a period of at least two weeks.
 19. The formulation of claim 18, wherein the peptide salt is Ac—D—Nal—D—pa—D—Pal—Ser—Tyr—D—Hci—Leu—Ilys—Pro—D—Ala—NH2trifluoroacetate.
 20. The formulation of claim 18, wherein the peptide salt is Ac—D—Nal—D—Cpa—D—Pal—Ser—Tyr—D—Hci—Leu—Ilys—Pro—D—Ala—NH₂ sulfate.
 21. The suspension of claim 1, wherein the suspension is a fluid, milky microcrystalline aqueous suspension.
 22. A lyophilized composition formed from a lyophilized suspension, which comprises the suspension of claim 21, wherein the peptide salt is Ac—D—Nal—D—Cpa—D—Pal—Ser—Tyr—D—Hci—Leu—Ilys—Pro—D—Ala—NH2trifluoroacetate, and the suspension further comprises an isotonic agent.
 23. A method of preparing a fluid, milky microcrystalline aqueous suspension of Ac—D—Nal—D—pa—D—Pal—Ser—Tyr—D—Hci—Leu—Ilys—Pro—D—Ala—NH₂trifluoroacetate comprising, adding water or buffer with mixing to the lyophilized composition of claim
 22. 